Real-time tracking of complex ubiquitination cascades using a fluorescent confocal on-bead assay

The ubiquitin-proteasome system (UPS) controls the stability, localization and/or activity of the proteome. However, the identification and characterization of complex individual ubiquitination cascades and their modulators remains a challenge. Here, we report a broadly applicable, multiplexed, miniaturized on-bead technique for real-time monitoring of various ubiquitination-related enzymatic activities. The assay, termed UPS-confocal fluorescence nanoscanning (UPS-CONA), employs a substrate of interest immobilized on a micro-bead and a fluorescently labelled ubiquitin which, upon enzymatic conjugation to the substrate, is quantitatively detected on the bead periphery by confocal imaging. UPS-CONA is suitable for studying individual enzymatic activities, including various E1, E2 and HECT-type E3 enzymes, and for monitoring multi-step reactions within ubiquitination cascades in a single experimental compartment. We demonstrate the power of the UPS-CONA technique by simultaneously following ubiquitin transfer from Ube1 through Ube2L3 to E6AP. We applied this multi-step setup to investigate the selectivity of five ubiquitination inhibitors reportedly targeting different classes of ubiquitination enzymes. Using UPS-CONA, we have identified a new activity of a small molecule E2 inhibitor, BAY 11-7082, and of a HECT E3 inhibitor, heclin, towards the Ube1 enzyme. As a sensitive, quantitative, flexible and reagent-efficient method with a straightforward protocol, UPS-CONA constitutes a powerful tool for interrogation of ubiquitination-related enzymatic pathways and their chemical modulators, and is readily scalable for large experiments.

Experimental Study on Effect of Heating Time on Polyurethane Foam Smoldering Propagation

Polyurethane foam is an important branch of building insulation materials, but its poor fire performance easily to lead to smoldering. This is conducted under condition of natural convection and heating time change of smoldering experiments on the smoldering experimental compartment. Use the thermocouple to measure the temperature changes inside the material. The results shows that, the heating time is shorter, smoldering into flame combustion is less complete, when time is short enough, smoldering extinguished; and in the vertical smoldering propagation, as time is shortened, smoldering into open fire’s position is upward. Experiment and analysis can be nicely displayed the effect of heating time on smoldering process.

Study on Material Parameters Effects on Smoldering and Transition from Smoldering to Flaming Combustion

The polyurethane foam is a most common fuel in smoldering fire. A small scale experimental compartment was built. The width, thickness and density of polyurethane foam material were changed several times in the experiments. Temperature histories measurement and analysis gaseous were used to explore the transition’s mechanism. The results show that with the width increased, the surface area and the air flow rate which access to the inside of material have also increased, and the quantity of oxygen is sufficient, heat release quantity by carbon oxidation is increased. When the width reaches a certain value, polyurethane foam material transformed easier from smoldering to flaming combustion. The polyurethane foam transforms to the flaming combustion not only depends on the length, width size but also relates to the thickness closely. When thickness reached a certain size, the temperature also reaches a whole jump. And the material transforms to the flaming combustion finally. The oxidation of larger density polyurethane foam material produces more heat, and then accumulates heat in the polyurethane foam center to reach the required temperature of gas phase reaction and ultimately transforms to flaming combustion from smoldering.

Effects of Exit Vent Location on Fire Room Conditions During PPV

Positive Pressure Ventilation (PPV) is a widely used fire fighting tactic in which a fan is used to push hot products of fire out of a burning structure. There is a recent body of research that has been conducted regarding the advantages and disadvantages of PPV. Studies of PPV most commonly use full scale experimental fires and/or computational simulations to evaluate its effectiveness. This paper presents computational simulations that have been conducted using Fire Dynamic Simulator (FDS) version 5 to evaluate the effects of exit vent location on resulting fire room conditions during the application of PPV to a ventilation constrained fire. The simulations use a simple one room structure with an adjacent hallway. We are simulating this geometry because we are in the process of designing and constructing a similar experimental compartment. Cold flow simulations are first conducted to understand how much the presence of the fire heat release affects the flow patterns. Then, two simulations which employ PPV with different exit vent locations are compared. The differences between the two simulations are detailed and a physical explanation for the differences is presented.